WO2015097460A1

WO2015097460A1 - Improved formulation

Info

Publication number: WO2015097460A1
Application number: PCT/GB2014/053814
Authority: WO
Inventors: Deirdre Patricia MCINTOSH; David John SHOTTON
Original assignee: Aimsco Limited
Priority date: 2013-12-23
Filing date: 2014-12-19
Publication date: 2015-07-02
Also published as: GB201322948D0

Abstract

The present invention relates to a CRH formulation having improved stability/ efficacy. The improved CRH formulation is particularly suitable for treatment of various disorders. The invention also relates to a method of producing the CRH formulation, and to methods of treatment using said CRH formulation.

Description

Improved formulation

The present invention relates to a formulation having improved stability/ efficacy. The formulation is particularly suitable for treatment of various disorders. The invention also relates to a method of producing the formulation, and to methods of treatment using said formulation.

WO 2006/021814 describes a serum composition comprising corticotropin releasing factor (CRF).

WO 2006/021814 also describes the use of CRF for treating a number of disorders, in particular multiple sclerosis and inflammatory disorders such as rheumatoid arthritis; optic neuritis; motor neuron disease; autoimmune diseases; axonal or nerve damage; and cancers. Particular cancers of interest include myelomas, melanomas and lymphomas. Other disorders include cardiovascular diseases; and neural disorders, both demyelinating and non-demyelinating. Examples of particular disorders which may be treated with CRF include cerebrovascular ischaemic disease; Alzheimer's disease; Huntingdon's chorea; mixed connective tissue diseases; scleroderma; anaphylaxis; septic shock; carditis and endocarditis; wound healing; contact dermatitis; occupational lung diseases; glomerulonephritis; transplant rejection; temporal arteritis; vasculitic diseases; hepatitis; and burns. Particular non-demyelinating disorders which may be treated include multiple system atrophy; epilepsy; muscular dystrophy; schizophrenia; bipolar disorder; depression; channelopathies; myasthenia gravis; pain due to malignant neoplasia; chronic fatigue syndrome; fibromyositis; irritable bowel syndrome; work related upper limb disorder; cluster headache; migraine; and chronic daily headache. Particular demyelinating disorders which may be treated include infections of the nervous system; nerve entrapment and focal injury; traumatic spinal cord injury; brachial plexopathy (idiopathic and traumatic, brachial neuritis, Parsonage Turner syndrome, neuralgic amyotrophy); radiculopathy; channelopathies; and tic douloureux. Particular autoimmune disorders which may be treated include lupus; psoriasis; eczema; thyroiditis; and polymyositis. Particular peripheral neuropathy of axonal and demyelinating type which may be treated include hereditary motor and sensor neuropathy of all types; Charcot-Marie-Tooth disease (CMT) types CMT1A, CMT1 B, CMT2, CMT3 (Dejerine Sottas disease), CMT4 (Types A, B, C and D), X-linked Charcot-Marie-Tooth disease (CMTX); Hereditary Neuropathy with liability to pressure palsies (HNPP), also called Tomaculous neuropathy; Hereditary Motor and Sensory Neuropathy with Deafness - Lom (HMSNL); Proximal Hereditary Motor and Sensory Neuropathy/Neuronopathy (HMSNP); Hereditary Neuralgic Amyotrophy; Hereditary Sensory and Autonomic Neuropathies (HSAN1 , HSAN2, HSAN3 (also called Riley-Day syndrome or familial dysautonomia), HSAN4, HSAN5); Familial Amyloid polyneuropathies (Type I, Type II, Type III, Type IV); Metachromatic Leukodystrophy; Krabbe's Disease; Fabry's Disease; Adrenoleukodystrophy; Refsum's disease (HMSN IV); Tangier Disease; Friedreich's ataxia; Spinal cerebellar ataxia (SCA) all types - SCA1 , SCA2, SCA3, SCA4, SCA5, SCA6, SCA7, SCA8, SCA10, SCA1 1 , SCA12, SCA13, SCA14, SCA16; Spinocerebellar Ataxia; Cockayne's syndrome and Giant axonal neuropathy. CRF is also identified as being useful in the treatment of chronic inflammatory demyelinating polyneuropathy (CIDP) and Guillain-Barre syndrome. CRF is also described as having anti-angiogenic properties, caused by the molecules thrombospondin-1 (TSP-1 ) and platelet factor-4 (PF-4). It is therefore understood that use of CRF, also known as corticotropin releasing hormone (CRH) also known as corticoliberin, is advantageous in treating the above- mentioned disorders in a patient.

One drawback associated with CRH formulations is that, following administration to a patient, the CRH protein has a limited effective biological half-life - by way of example, the CRH protein has a very low effective plasma half-life (approximately 4 minutes), and thus a low bioavailability/ efficacy, relying on a pulsatile stimulus.

Attempts to achieve long-term persistent levels of CRH in vivo have been reported, e.g. by Walker JJ et al (Walker et al (2012) "The origin of glucocorticoid hormone oscillations; PLoS Biol 10(6):e1001341 ). Even with constant infusion of CRH over a six-hour period, however, elevated levels of CRH can be maintained in vivo for no longer than an hour before falling back toward base-line levels; this effect appears to persist regardless of the dosage used. Moreover, constant infusion of CRH is undesirable and an impractical therapeutic method outside of a hospital or laboratory setting. There is therefore a need for a CRH formulation having a longer effective biological half-life and thus a longer effective therapeutic window of efficacy.

Another drawback associated with CRH formulations is that said formulations have limited stability, and thus sub-optimal efficacy. There is therefore a need for a CRH formulation having improved stability and thus a longer effective therapeutic window of efficacy.

The present invention addresses one or more of the above needs by providing a stabilised CRH formulation, which demonstrates an extended effective biological half-life (e.g. persistence) in the body. Therapeutic uses of such stabilised CRH are also provided for the prevention or treatment of one or more diseases (including, but not limited to, said hereinbefore listed disorders).

In more detail, the present invention provides a formulation comprising a stabilised complex of CRH and alpha-2 macroglobulin. The present inventors believe that the formation of said complex (having an approximate molecular weight of 800 kDa) is responsible for improved stability and efficacy of the formulation.

The alpha-2 macroglobulin is present in the formulation of the invention at a concentration of more than 150,000 picograms per ml for example more than 160,000 picograms per ml, or more than 170,000 picograms per ml, or more than 180,000 picograms per ml, or more than 190,000 picograms per mi, or more than 200,000 picograms per ml, or more than 210,000 picograms per ml, or more than 220,000 picograms per ml, or more than 230,000 picograms per ml, or more than 250,000 picograms per ml. Thus, the present invention provides alpha-2 macroglobulin in a preferred amount for complexing with CRH. In one embodiment, alpha-2 macroglobulin is present in the formulation of the invention at a concentration of at least 1 μΜ (micro molar), at least 1 .5 μΜ, at least 2 μΜ, typically in the range 2-5 μΜ.

Similarly, the CRH is present in the formulation of the invention at a concentration of more than 120 pg/ml, for example more than 130 pg/ml, or more than 140 pg/ml, or more than 150 pg/ml, or more than 160 pg/ml, or more than 170 pg/ml, or more than 180 pg/ml, or more than 200 pg/ml. Thus, the present invention provides CRH in an amount that is preferred for complexing with alpha-2 macroglobulin. Lower concentration of CRH are possible though less preferred, such as more than 80 pg/ml, for example more than 90 pg/ml, more than 100 pg/ml, or more than 1 10 pg/ml.

The alpha-2 macroglobulin may be present in the formulation of the invention at a concentration of more than 150,000 picograms per millilitre, and the CRH may be present at a concentration of more than 80 pg/ml. The alpha-2 macroglobulin may be present in the formulation of the invention at a concentration of more thanl 50,000 pg/ml (or more than 1 micro molar), and the CRH may be present at a concentration of more than 100 pg/ml (such as more than 120 pg/ml). The formulation of the invention may further comprise CRH binding protein (CRH- BP). The CRH-BP may be present at a concentration of more than 60 pg/ml, for example more than 70 pg/ml, or more than 70 pg/ml, or more than 80 pg/ml, or more than 90 pg/ml, or more than 100 pg/ml, or more than 120 pg/ml, or more than 150 pg/ml. Lower concentrations of CRH-BP may be employed such as more than 40 or more than 50 pg/ml. A typical upper range for CRH-BP would be in the region of 300 pg/ml.

The present invention also provides a method of manufacturing a stabilised complex of CRH and alpha-2 macroglobulin, the method comprising:

(a) providing hyperimmune serum from an ungulate that has been immunised with an immunodeficiency virus; and (b) aliquoting said serum into a vial, wherein said serum is in a form for administration to a patient and comprises the stabilised complex of CRH and alpha-2 macroglobulin;

wherein the serum remains unfrozen throughout the method up to and including the aliquoting step,

and wherein said method retains molecules having a size of greater than 0.2 microns (or greater than 200 kDa).

Thus, the manufacture method of the present invention proactively retains molecules having a size greater than 0.2 microns (or greater than 200 kDa). In one embodiment, the method does not include a 0.2 micron pore size filtration step. For example, the method does not include any filtration step employing a filter pore size of 0.2 microns or less that removes molecules having a size greater than 0.2 microns (or greater than 200 kDa). Thus, the method proactively retains large molecules (e.g. alpha-2 macroglobulin, and preferably stabilized 'CRH - alpha-2 macroglobulin' complex though the latter may re-associate post-filtration).

The manufacture method of the invention may include an optional step of treating the serum prior to aliquoting. By way of example, a suitable precipitation step may be introduced, such as (conventional) ammonium sulphate precipitation. This may be followed by resuspension of the precipitate in an aqueous solution (e.g. PBS buffer), and dialysis - by way of example, a 10 kDa membrane cut-off diafiltration step may be employed (thereby retaining all molecules having a molecular weight greater than 10 kDa). Alternate means known to a person skilled in the art (e.g. separation columns, etc.) may equally be employed.

The manufacture method of the invention may include an optional microfiltration step (to remove large macromolecules and any other undesirable large structures) whilst retaining molecules having a size (e.g. average diameter) greater than 0.2 microns (or greater than 200 kDa), which pass through the filter as filtrate. A microfiltration step may be included in combination with or separate from any 'treating of serum' step (described above). If employed, a microfiltration step is performed before the 'aliquoting' step (and after any 'treating of serum' step, if present). In one embodiment, molecules having a size greater than 0.3 microns (or greater than 300kDa) are retained (e.g. by employing a 0.3 micron pore size microfilter). An alternative microfiltration step may be employed to retain molecules having a size greater than 0.4 microns (or greater than 400 kDa), or to retain molecules having a size greater than 0.5 microns (or greater than 500 kDa), or to retain molecules having a size greater than 0.6 microns (or greater than 600 kDa), or to retain molecules having a size greater than 0.7 microns (or greater than 700 kDa), or to retain molecules having a size greater than 0.75 microns (or greater than 750 kDa), or to retain molecules having a size greater than 0.8 microns (or greater than 800 kDa), or to retain molecules having a size greater than 0.85 microns (or greater than 850 kDa), or to retain molecules having a size greater than 0.9 microns (or greater than 900 kDa). By employing a high microfilter (such as a large pore size filter as hereinbefore described, for example greater than 0.2 microns, such as in the range of 0.3 to 0.9 microns), the present inventors believe that stabilised complex (i.e. 'CRH - alpha-2 macroglobulin' complex) in an optimal form and/ or stabilised complex having improved efficacy is presented in the formulation of the present invention. At the same time, any undesirable macromolecules having a size greater than the defined pore size are captured by the microfilter. In this regard, microfiltration to retain molecules in the filtrate having a molecular weight of more than 800 kDa or more than 850 kDa (e.g. employing a filter pore size of at least Ο.δμιη filter, or at least 0.85μιη) is desirable.

The manufacture method of the invention includes an aliquoting step in which the serum is aliquoted into vials, optionally with protein concentration adjustment, to provide a single dose amount. At this stage it may be frozen (e.g. at minus 22 degrees C) prior to use. In this regard, prior to use, the aliquoted serum is thawed, followed by prompt administration (e.g. within 6 hours, preferably within 4 hours, preferably within 1 hour, preferably within 5 minutes, preferably within 1 minute) to a patient.

In one embodiment, the stabilised CRH formulation of the present invention is prepared by a method that comprises: providing isolated blood from an ungulate (e.g. a goat), wherein said ungulate has been immunised with an autoimmune virus (optionally attenuated or otherwise inactivated), and obtaining serum from the blood (e.g. by centrifugation); treating the serum to separate the CRH and other active components of interest; diafiltration (e.g. using a l OkDa cut off membrane) of the separated serum comprising CRH and other active components of interest, thereby retaining molecules having a molecular weight of at least 10 kDa; wherein all of the above steps are performed under cooled conditions (e.g. less than 22 degrees C, such as less than 10 degrees C, or less than 5 degrees C); whilst ensuring that the serum remains unfrozen following treatment to separate the CRH and other active components.

Thus, the basic methodology as described in WO 2006/021814, which is hereby incorporated in its entirety by reference thereto, may be employed, though with the inclusion of one or more additional steps selected from (i) specific retention of molecules having a size greater than 0.2 microns (greater than 200 kDa); (ii) avoidance of multiple freeze-thaw steps and/ or minimising ambient temperature exposure; and/ or (iii) a mixing/ agitation step (to enhance CRH: alpha-2 macroglobulin complex formation).

The autoimmune virus may be HIV or SIV, which may be HIV 1MB; the autoimmune virus may be in the form of a lysate or a heat-killed virus.

In this regard, "serum" is defined as that component of the blood from which the blood cells have been removed, e.g. by centrifugation. Optionally, exposure to ambient temperature (e.g. room temperature, 22 degrees C) is strictly minimised at all stages of the method - by way of example, cold trays (ensuring a maximum temperature of less than 22 degrees C, or less than 10 degrees C, or less than 7 degrees C, or less than 5 degrees C) and other such means are used throughout. Thus the composition may be kept at a constant temperature below 22 degrees C, e.g. at less than 10 degrees C, or less than 7 degrees C, or less than 5 degrees C. It is preferred that the method is carried out as a continuous process, avoiding any freezing steps prior to storage. For example, no freezing step is employed prior to final aliquoting into vials (optionally with adjustment of protein concentration). In particular, it is preferred that no freezing step is carried out once the serum has been treated to separate the CRH and other components of interest. One or more agitation steps may be carried out during the method; these agitation steps may use cold trays.

The manufacture method may comprise a final step (e.g. after any protein concentration adjustment step) of freezing for subsequent storage. In one embodiment, no freezing step is employed prior to protein adjustment, or aliquoting.

Without wishing to be bound by any theory, the present inventors believe that the bioactive molecules within the composition comprising CRH are prone to undesirable aggregation upon freezing and thus this step should not be performed more than once prior to use. Thus, multiple freezing-thawing steps should be avoided as they are believed to result in inactivation of key bioactive molecules within the CRH composition and/or lead to removal thereof during the manufacture method.

Alternatively, the CRH formulation of the present invention may be prepared from first principles based on commercially available components (including recombinantly prepared components).

The two principal components of the herein described formulation (when present in the stabilised complex form) may have a ratio of 4: 1 CRH:alpha-2 macroglobulin, 2: 1 CRH:alpha-2 macroglobulin, 1 : 1 CRH:alpha-2 macroglobulin, or combinations thereof. In one embodiment, the predominant complexed form of CRH: alpha-2 macroglobulin is a macromolecular quaternary complex (i.e. 4: 1 ).

The CRH and alpha-2 macroglobulin components may be complexed together via non-covalent bonds such as one or more of hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions. The above-described concentrations typically refer to the concentrations obtained during the manufacture process such as immediately prior aliquoting and optional freezing for subsequent storage (optionally including any protein concentration adjustment) that yields the ready-to-use formulation (typically having a concentration of 4-5 mg protein per ml).

The CRH may be human or non-human. For example, the CRH may be an ungulate CRH such as horse, zebra, donkey, cattle, bison, goat, pig, moose, elk, deer, antelope or gazelle. In one embodiment the CRH is not ovine CRH. In a most preferred embodiment, the CRH is caprine CRH.

Corticotropin releasing hormone (CRH), also known as corticoliberin, is a 41 residue peptide originally isolated from ovine hypothalamus based on its ability to stimulate the hypothalamic-pituitary adrenal axis from cultured anterior pituitary cells. CRH is the principal neuroregulator of the basal and stress-induced secretion of ACTH, β- endorphin, and other pro-opiomelanocortin related peptides from the paraventricular nucleus of the anterior pituitary gland. In addition to its endocrine function, mediation of CRH specific responses occur via its cognate receptors (they include CRH-R1 , CRH-R2a, CRH-R2P, CRH-R2y and CRH-binding protein [CRH-BP]). CRH-R1 is a 415 amino acid protein that shows sequence homology across different species (human, mouse and rat). CRH-binding protein represents the smallest receptor at 322 amino acids, and acts as an inhibitor of free CRH. CRH-R1 and CRH-R2 are both ubiquitously expressed on the cell surface of the hypothalamus, cerebellum, cortex, amygdala, subcortex, immune cells, gut and skin. Conversely, CRH-BP is found predominantly in the liver, placenta and brain. Importantly there appears to be no significant overlap in distribution of the said receptors. This likely reflects differing functional roles. An example of this is seen during pregnancy where elevation in peripheral CRH is regulated by an elevation in secreted levels of CRH binding protein. The overall effect of this is to prevent an elevation in peripheral circulating levels of glucocorticoids during pregnancy. Several forms of CRH have been identified in nature, they include a high molecular weight form 194 amino acids Mw - 30,000, a Mw -18,000, Mw -7,500 and the 41 amino acid residue. All three forms are biologically active and able to stimulate ACTH release.

Alpha-2-macroglobulin, also known as A2M, is a large plasma protein found in blood. It is produced by the liver and is the largest major non-immunoglobulin protein in plasma. A2M is synthesized primarily by the liver and is also produced locally by macrophages, fibroblasts and adrenocortical cells. Alpha-2-macroglobulin acts as an anti-protease and is able to inactivate an enormous variety of proteinases. It also functions as a carrier protein binding to numerous growth factors and cytokines. Examples include transferrin (where A2M regulates the binding of to the surface receptor), binds defensin and myelin basic protein, binds several important cytokines, including basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), nerve growth factor (NGF), interleukin-1 β (IL-Ι β) and interleukin-6 (IL-6), transforming growth factor (TGF-Ι β), and insulin, and modify their biological activity. Human A2M is composed of four identical subunits bound together by disulphide-S-S- bonds. The principal mechanism by which A2M inhibits proteases is through steric hindrance. The mechanism involves protease cleavage of the thiol 35 amino acid bait region, a segment of the molecule, which is particularly susceptible to proteolytic cleavage, which initiates conformational change such that A2M collapses about the protease thus resulting in its inhibition. In the resulting A2M- protease complex, the active site of the protease is sterically shielded, thus substantially decreasing access to protein substrates including those that are bound to active A2M. Decreases in A2M have been associated with a variety of diseases, for example a common variant (29.5%) polymorphism of A2M leads to an increase in the risk of Alzheimer's disease.

Stabilisation of CRH by binding to alpha-2 macroglobulin, and the attendant enhanced effective biological half-life is one effect of formulations of the present invention. Said stability provides an enhanced (longer) plasma half-life - by way of example, the stabilised complex of the present invention has a half-life of at least 24 hours. The stabilised complex of the invention may be employed in combination with one or more other stabilisers, such as fibronectin or albumin. Additionally or alternatively, the stabilised complex of the invention may be employed in combination with a proopiomelanocortin (POMC) peptide. The use of POMC peptides in this manner stimulates further in vivo POMC production, and/ or helps to induce a host response before endogenous POMC peptide levels are stimulated.

POMC may be present in the formulation at a concentration in the range of between 100 picomoles per litre (pmol/l) and 200 pmol/l.

Thus, in one embodiment the invention provides a stabilised CRH formulation having component proportions as defined hereinbefore. For example, the formulation may have between 150,000 and 5000,000 (e.g. at least 250,000) picograms per millilitre (pg/ml) of alpha-2 macroglobulin, between 100 pg/ml and 2000 (e.g. at least 120 or 150) pg/ml of CRH, and optionally between 140 pmol/l and 180 (e.g. at least 160) pmol/l of POMC.

Without wishing to be bound by any theory, the present inventors believe that alpha- 2 macroglobulin inhibits subtilisin serine endopeptidases (pro-hormone convertases), which may otherwise exert deleterious effects on POMC prior to administration.

Following administration, in vivo activation of POMC may occur by the direct actions of prohormone convertase 1 (PC1 ), prohormone convertase 2 (PC2), carboxypeptidase E (CPE), peptidyl alpha-amidating monooxygenase (PAM), N- acetyltransferase (N-AT) and prolylcarboxypeptidase (PRCP) acting in a tissue specific manner. All said POMC cleavage sites appear to be acted upon by proteases in the hypothalamus, placenta, epithelium and leucocytes.

Human POMC peptide is described in detail in entry 176830 of OMIM (online mendelian inheritance in man, accessible through http://www.ncbi. nlm.nih.qovA). The nucleotide and amino acid sequence of human POMC is also known, and has GENBANK accession number BC065832. Human POMC gives rise to a glycosylated protein precursor having a molecular weight of 31 kDa. By a "POMC peptide" is meant any peptide having a corresponding sequence, structure, or function. It will be apparent to the skilled person that the canonical nucleotide and/or amino acid sequences given for human POMC in the GENBANK entry referenced above may be varied to a certain degree without affecting the structure or function of the peptide. In particular, allelic variants and functional mutants are included within this definition. Mutants may include conservative amino acid substitutions. A "POMC peptide" as used herein refers to any peptide acting as a precursor to at least one form of MSH, ACTH, at least one form of lipotrophin (LPH), β endorphin, met-enkephalin and leu-enkephalin; and preferably all of α, β, and γ MSH; ACTH; β and γ LPH; and β endorphin, met- enkephalin and leu- enkephalin.

The POMC peptide may be human or non-human POMC. In one embodiment the POMC peptide is an ungulate POMC such as horse, zebra, donkey, cattle, bison, goat, pig, moose, elk, deer, antelope or gazelle. In one embodiment the POMC peptide is not a rodent (e.g. mouse or rat) POMC peptide.

Administration of a POMC peptide has a self-sustaining effect, in that administration of an initial amount of POMC peptide leads to endogenous production of POMC in the patient; thus, an initial administration of a low level of POMC has a significant effect on the patient.

The formulation of the invention selectively increases the enzymatic degradation of POMC in vivo. The formulation of the invention increases the release of POMC- derived peptides such as ACTH, alpha-MSH, beta-MSH, CLIP, Lipotrophin-gamma, met-enkephalins and beta-endorphins. Longer-term administration of the formulation typically leads to a sustainable increase in POMC-derived peptides in a patient. The stabilised complex of the invention acts to enhance the central CRH-1 regulatory response. This results in optimisation of key anti-inflammatory cytokines and abrogation of certain pro-inflammatory cytokines related to the Th1 -mediated response.

Administration of CRH to a patient stimulates production of endogenous CRH and thus leads to a self-sustaining effect. CRH can therefore be administered at a low concentration to a patient.

The stabilised complex of the invention also protects CRH from proteolytic degradation by proteases and accordingly administration of the stabilised complex of the invention provides slow release of CRH in the circulation and a significant increase in CRH levels.

Thus, the stabilised complex of the invention leads to persistent, elevated levels of CRH in vivo for at least 12 hours following administration, for at least 24 hours following administration, or for at least 48 hours following administration. Administration of the stabilised complex of the invention may lead to an in vivo increase in CRH concentration of between 25% and 50% from patient baseline CRH levels at 24 hours from administration, and an in vivo increase of between 75% and 100% from patient baseline CRH levels at 48 hours from administration.

This contrasts with administration of formulations containing CRH in similar concentrations where the CRH is not in a stabilised complex (or in a poorer stabilised complex including, for example CRH aggregates): an in vivo increase in CRH concentration from patient baseline CRH levels may be seen within 24 hours of administration, but the effect does not persist and within 36 hours from administration CRH concentration is falling.

The formulation of the invention selectively up-regulates the CRH-1 receptor both centrally and peripherally in key target issues and enhances the central CRH-1 regulatory response, while also leading to selective down-regulation of CRH-2 specific receptors and up-regulation of CRH-binding protein in tissues such as the adrenal cortex. It is believed by the inventors that administration of the formulation will thus lead to optimisation of key inflammatory cytokines and down-regulation of certain pro-inflammatory cytokines related to the Th-1 mediated response, through targeting leucocytes and macrophages.

No secondary glucocorticoid surge from increased levels of ACTH are noted despite the elevated CRH levels, in part due to further processing and cleavage of ACTH and a reduction in "free" CRH levels. Elevated "free" peripheral CRH may also be regulated by a parallel increase in CRH-BP. This again mitigates excessive production of unwanted glucocorticoids. The combination of the peripheral and central dynamic relationship provides a prolonged activation of the CRH pathway without negative feedback. Central CRH is then able to further target T-helper immune cells and macrophages that express CRH.

The combined net effect of all of the above is an anti-inflammatory, reparative and fundamental immunomodulatory therapeutic that works within homeostatic constraints. The unique targeting and accessibility of the stabilised complex of the invention explains its versatility in a wide range of diseases.

Thus the present invention provides a stabilised CRH formulation that works within homeostatic constraints following in vivo administration.

The present invention accordingly further provides a formulation for use in prevention or treatment of one or more diseases selected from systemic sclerosis (SSc), multiple sclerosis; inflammatory disorders such as rheumatoid arthritis, osteoarthritis, inflammatory tendonopathies, inflammatory bowel disease and inflammation of the lung, including emphysema, lung fibrosis, alveolitis and cystic fibrosis; optic neuritis; motor neuron disease; autoimmune diseases; axonal or nerve damage; and cancers (including myelomas, melanomas and lymphomas); cardiovascular diseases; neural disorders, both demyelinating and non-demyelinating; cerebrovascular ischemic disease; Alzheimer's disease; Parkinson's disease; Huntingdon's chorea; mixed connective tissue diseases; scleroderma; anaphylaxis; septic shock; endotoxaemia; carditis and endocarditis; wound healing; contact dermatitis; occupational lung diseases; glomerulonephritis; transplant rejection; temporal arteritis; vasculitic diseases; hepatitis (in particular hepatitis C); burns; multiple system atrophy; epilepsy; muscular dystrophy; schizophrenia; bipolar disorder; depression; channelopathies; myasthenia gravis; pain due to malignant neoplasia; chronic fatigue syndrome; fibromyositis; irritable bowel syndrome; work related upper limb disorder; cluster headache; migraine; chronic daily headache; infections of the nervous system; nerve entrapment and focal injury; traumatic spinal cord injury; brachial plexopathy (idiopathic and traumatic, brachial neuritis, Parsonage Turner syndrome, neuralgic amyotrophy); radiculopathy; channelopathies; tic douloureux; lupus; psoriasis; eczema; thyroiditis; polymysotis; hereditary motor and sensor neuropathy of all types; Charcot-Marie-Tooth disease (CMT) types CMT1A, CMT1 B, CMT2, CMT3 (Dejerine Sottas disease), CMT4 (Types A, B, C and D), X-linked Charcot-Marie-Tooth disease (CMTX); Hereditary Neuropathy with liability to pressure palsies (HNPP), also called Tomaculous neuropathy; Hereditary Motor and Sensory Neuropathy with Deafness - Lorn (HMSNL); Proximal Hereditary Motor and Sensory Neuropathy/Neuronopathy (HMSNP); Hereditary Neuralgic Amyotrophy; Hereditary Sensory and Autonomic Neuropathies (HSAN1 , HSAN2, HSAN3 (also called Riley-Day syndrome or familial dysautonomia), HSAN4, HSAN5); Familial Amyloid polyneuropathies (Type I, Type II, Type III, Type IV); Metachromatic Leukodystrophy; Krabbe's Disease; Fabry's Disease; Adrenoleukodystrophy; Refsum's disease (HMSN IV); Tangier Disease; Friedreich's ataxia; Spinal cerebellar ataxia (SCA) all types - SCA1 , SCA2, SCA3, SCA4, SCA5, SCA6, SCA7, SCA8, SCA10, SCA1 1 , SCA12, SCA13, SCA14, SCA16; Spinocerebellar Ataxia; Cockayne's syndrome; Giant axonal neuropathy; chronic inflammatory demyelinating polyneuropathy (CIDP); and Guillain-Barre syndrome. The formulation of the present invention may take the form of a pharmaceutical composition. The invention accordingly provides a pharmaceutical composition comprising the stabilised complex of the invention, and the use thereof in preventing or treating of one or more of the above-mentioned diseases. Administration of the formulation of the invention may be accomplished orally or parenterally. Methods of parenteral delivery include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intra-ventricular, intravenous, intraperitoneal, or intranasal administration. In addition to the active ingredients, the formulation of the invention may comprise suitable pharmaceutically acceptable carriers comprising excipients and other components which facilitate processing of the active compounds into preparations suitable for pharmaceutical administration.

Oral formulations may include pharmaceutically acceptable carriers known in the art in dosages suitable for oral administration. Such carriers enable the compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like suitable for ingestion by the subject.

Formulation for oral use can be obtained through combination of active compounds with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds if desired to obtain tablets or dragee cores. Suitable excipients include carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methylceilulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen. If desired, disintegrating or solubilising agents may be added, such as cross linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof.

Dragee cores can be provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterise the quantity of active compound. Formulations for oral use include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally stabilisers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilisers. Formulations for parenteral administration include aqueous solutions of active compounds. For injection, the formulations of the invention may take the form of aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous suspension injections can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension can also contain suitable stabilisers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated may be used in the formulation.

The formulations of the present invention can be manufactured substantially in accordance with standard manufacturing procedures known in the art. The formulation may also comprise one or more peptide regulatory or releasing factors, which may induce a cascade of release of further peptides by a variety of cells in the patient. Such additional factors are typically provided from the same animal species as the CRH. Suitable factors include a- HLA, TGF-β, and IL-10, among others.

In one embodiment, the formulation may comprise one or more of vasopressin, beta endorphin, and an enkephalin. In certain embodiments, the formulation may comprise CRH binding protein, CRH-BP. This binds CRH and acts as a reservoir for subsequent release of CRH to the patient. The present invention also provides a method of treatment for a disease selected from systemic sclerosis (SSc); multiple sclerosis; rheumatoid arthritis; optic neuritis; Parkinson's disease; motor neuron disease; autoimmune diseases including lupus, psoriasis, eczema, thyroiditis, and polymyositis; axonal or nerve damage; cancers, in particular myelomas, melanomas, and lymphomas; neural disorders, both demyelinating and non-demyelinating; inflammatory conditions; obesity; nerve conduction disorders; and sexual dysfunction, in particular erectile dysfunction; the method comprising administering the formulation of the invention to a patient in need thereof.

The optimal dosage will be determined by the clinician. For example, administration may be in a dosage of between 0.01 and 10 mg (total protein) per kg (patient), for example between 0.01 and 5 mg/kg, between 0.025 and 2 mg/kg, or between 0.05 and 1 mg/kg. A product suitable for administration to patients may have a total protein concentration of approximately 4 mg/ml. Improved patient responses have been observed when a staged dosage regimen is employed, for example, based on initial 0.1 ml administrations, followed by 0.5ml administrations, followed by 1 ml administrations. The precise dosage to be administered may be varied depending on such factors as the age, sex and weight of the patient, the method and formulation of administration, as well as the nature and severity of the disorder to be treated. Other factors such as diet, time of administration, condition of the patient, drug combinations, and reaction sensitivity may be taken into account. An effective treatment regimen may be determined by the clinician responsible for the treatment. One or more administrations may be given, and typically the benefits are observed after a series of at least three, five, or more administrations. Repeated administration may be desirable to maintain the beneficial effects of the composition. The treatment may be administered by any effective route, such as by subcutaneous injection, although alternative routes which may be used include intramuscular or intra-lesional injection, oral, aerosol, parenteral, topical or via a suppository. The treatment may be administered as a liquid formulation, although other formulations may be used. For example, the treatment may be mixed with suitable pharmaceutically acceptable carriers, and may be formulated as solids (tablets, pills, capsules, granules, etc) in a suitable composition for oral, topical or parenteral administration.

The invention also provides use of the aforementioned formulation in the preparation of a medicament for the treatment of one or more of the diseases recited above.

Figures

These and other aspects of the present invention will now be described by way of example only with reference to the accompanying figures.

Figure 1 illustrates the effect of introducing a small pore size filtration step during the manufacture protocol. The resulting concentration of a range of anti-inflammatory cytokines (IL-10, IL-6, IL-1 beta, and IFN gamma) and CRH (also referred to herein as CRF) were monitored both before and immediately after a 10nm (20N) or 35nm (35N) filtration step. For each molecule monitored, the first two columns correspond to before (unfiltered) and after (filtered) a 20 nm filtration step. Similarly, for each molecule monitored, the last two columns correspond to before (unfiltered) and after (filtered) a 35 nm filtration step. Two significant observations can be made by comparing the 'before' and 'after' filtration steps. First, filtration removes otherwise advantageous anti-inflammatory cytokines. Secondly, referring to the CRH data set, filtration reduces the concentration of this key therapeutic component of the present invention.

Figures 2-7 (and Table 1 ) illustrate data generated via the methodology described in Example 2. In more detail, these Figures demonstrate the in vivo concentration of CRH following administration of 3 different formulations, namely a formulation of the present invention is illustrated as "Aimspro+". Prior art formulations as per WO 2003/004049, WO 2003/064472, WO 2005/056053, WO 2005/097183, WO 2006/021814, and WO 2007/077465 are illustrated as "Aimspro A". The data include two 'control' formulations based on naive serum (designated "N35" & "Naive"). The y-axes of the graphs in Figures 2 to 7 give the CRH concentration in pg/ml. Figures 8-1 1 (and Table 2) illustrate data generated via the methodology described in Example 2. In more detail, these Figures demonstrate the corresponding in vivo concentration of CRH-BP. The y-axes of the graphs in Figures 8 to 1 1 give the CRH- BP concentration in pg/ml. The data confirm that all CRH-BP concentrations are within a physiological acceptable range.

Figures 12-14 illustrate data generated via the methodology described in Example 2. In more detail, these Figures demonstrate the corresponding concentration of alpha- 2 macroglobulin. The y-axes of the graphs in Figures 12 to 14 give the alpha-2 macroglobulin concentration in pg/ml. The formulation of the present invention ("Aimspro+") demonstrates the highest initial (0 hours) concentration of alpha-2 macroglobulin (Figure 14), which is significantly greater than the initial concentration observed with the prior art "Aimspro A" formulations (Figure 13). The high initial alpha-2 macroglobulin concentration is consistent with the proactive approach described herein to maximise retention of alpha-2 macroglobulin (and thus 'alpha-2 macroglobulin - CRH' stabilised complex). Moreover, even when compared with Applicant's modified formulation "Aimspro" (Figure 12), the present invention provides a formulation ("Aimspro+") that is significantly more stable (i.e. longer half- life and greater area under the curve) than "Aimspro". Examples

CRH and products containing CRH are very sensitive to proteolytic degradation and suffer from the above-mentioned poor effective half-life following in vivo administration. We show here that the stabilized complex of the invention has improved in vivo stability and, in view of said enhanced biological half-life, is able to demonstrate a greater therapeutic efficacy.

Example 1: Manufacture of the stabilised complex of the invention Hyperimmune ungulate serum is centrifuged to separate any unwanted components, and the method carried out as a continuous process, avoiding any freezing or thawing step(s) prior to final aliquoting. This avoids any aggregation and loss of the CRH component from the formulation.

In more detail, a serum composition comprising CRH was stored at 2 to 8 degrees C (and not frozen) and was diluted at a ratio of 1 :2 parts serum:cold PBS, and supersaturated ammonium sulphate was added slowly with constant agitation until a ratio of 47:53 of ammonium sulphate: PBS was reached. This was carried out on a cold tray and the resulting solution was maintained at this temperature for 30 to 60 minutes with constant agitation.

The serum solution was then centrifuged in a Beckman J6M/E centrifuge at 3500 rpm for 45 minutes at 4 degrees C. The supernatant was removed and discarded. The precipitated solid material was re-suspended in cold 47% saturated ammonium sulphate: PBS solution and re-centrifuged at 3500 rpm at 4 degrees C for 45 minutes. The supernatant was again discarded and the precipitated solid material re- suspended in ice cold PBS buffer. This solution (the serum component) was then subjected to diafiltration at 4 degrees C against PBS with a molecular weight cut-off of 10,000 Daltons.

The solution was adjusted to a protein concentration of between 4 to 5 milligrams per millilitre with ice-cold PBS. Small batches of the solution (1 .2 millilitres) containing the stabilised composition of the invention were put into vials in an isolator. 1 millilitre single doses were thus obtained and stored at -15 to -25 degrees C prior to use.

All steps were carried out as a continuous process in the cold except filling into vials, which was conducted so as to minimise exposure to ambient temperature at all times. Cold trays were used whenever possible. Following treatment of the serum with PBS and supersaturated ammonium sulphate, freezing was avoided until the final filling stage and subsequent storage. Example 2: The stabilised complex provides a persistent, elevated concentration of CRH in vivo

The stabilised complex of the invention has been compared with prior art formulations as previously disclosed by applicant.

Applicant has disclosed the same basic manufacture protocols in WO 2003/004049, WO 2003/064472, WO 2005/056053, WO 2005/097183, WO 2006/021814, and WO 2007/077465. The formulation of these prior art teachings is referred to herein as "Aimspro A".

In this Example, male mice, C57BL/6, -25 gm were divided into three groups (n=6 animals per group and time point measured):

1 ) one group was administered the stabilised complex of the invention (designated "Aimspro+");

2) another group was administered a composition comprising a CRH formulation prepared by Applicant's prior art basic manufacture protocol (designated "Aimspro A");

3) a third group was administered a composition comprising a CRH formulation prepared by Applicant's modified manufacture protocol employing a 0.2 micron microfiltration step followed by a 35 nm nanofiltration step (designated "Aimspro").

Two 'control' formulations were also employed:

1 ) a naive caprine serum (i.e. from a goat that had not been immunised) but which had been otherwise prepared by exactly the same manufacture method as per protocol 3) above (designated "N35"); and

2) a naive caprine serum (i.e. from a goat that had not been immunised) but which had been otherwise prepared without any 35 nm filtration step (designated "Naive").

Samples of approximately 250μΙ of whole blood (retro-orbital) were collected via the retro-orbital plexus using a microcapillary (EDTA) tube at intervals of 15 minutes, half an hour, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours and 48 hours from initial administration. Lithium Heparin-whole blood was centrifuged at 4 degrees C for 10 minutes at a RCF of 1000 and stored at -80 degrees C until CRH determination was carried out by ELISA (each in triplicate per animal per time point measured).

The average CRH concentration was calculated in respect of each population and the results are shown in Figures 2-7 (plus Table 1 ).

It can be seen from Figure 4 that the composition of the invention (labelled "Aimspro+") provided a steady, sustained increase in CRH concentration within the population over time. In particular, compared with "Aimspro" (Figure 2) a more rapid onset of high CRH concentration (greater than 20 pg/ml) was achieved at the 20 hour stage, which was maintained at the 48 hour stage. Similarly, compared with "Aimspro A" (Figure 3), no noticeable drop-off of CRH concentration was observed at the 48 hour stage.

By contrast, the "N35" naive filtered serum (Figure 5) and the unfiltered "Naive" serum (Figure 6) did not provide any sustained expression profile and the level of CRH (even after the 20 hour stage) was clearly falling back to initial levels.

The CRH data are summarised immediately below in Table 1 .

Table 1

By way of comparison, the level of CRH binding protein (CRH-BP) was also measured and the average result was calculated for each population over time. The results are shown in Figures 8-1 1 (and Table 2). It can be seen that the

profile is similar in each population.

The CRH-BP data are summarised immediately below in Table 2.

Table 2

As part of the same experiments, the concentration of alpha-2 macroglobulin was also monitored (see Figures 12-14). In summary, the "Aimspro+" formulation of the present invention (Figure 14) demonstrated the highest initial in vivo concentration of alpha-2 macroglobulin AND the longest half-life thereof (area under the curve). These data collectively support the understanding of the present inventors that the manufacture protocol of the present invention achieves an improved formulation in which the key biologically active component (stabilised 'alpha-2 macroglobulin - CRH' complex) is provided in an optimal form and thus preserved in this form for clinical use.

Claims

A method of manufacturing a stabilised complex of CRH and alpha-2 macroglobulin, the method comprising:

(a) providing hyperimmune serum from an ungulate that has been immunised with an immunodeficiency virus; and

(b) aliquoting said serum into a vial, wherein said serum is in a form for administration to a patient and comprises the stabilised complex of CRH and alpha-2 macroglobulin;

The method of claim 1 , wherein said method does not include a filtration step employing a filter pore size of 0.2 microns or less that removes molecules having a size greater than 0.2 microns (or greater than 200 kDa).

The method of claim 1 or claim 2, wherein a microfiltration step is introduced between steps a) and b),

and wherein molecules having a size greater than 0.3 microns (or greater than 300 kDa) are retained;

preferably wherein molecules having a size greater than 0.8 microns (or greater than 800 kDa) are retained.

The method of any preceding claim, wherein the serum is frozen after step (b) and thawed prior to patient administration, though with the proviso that said serum is not subjected to a subsequent freezing step prior to said patient administration.

The method of any one of claims 1 to 4, further comprising the step of adjusting the final protein concentration to 4-5 milligrams protein per millilitre; preferably wherein the hyperimmune serum is only frozen after said adjusting of the final protein concentration.

6. The method of any one of claims 1 to 5, wherein exposure to ambient temperature is strictly minimised at all stages of the method.

7 A formulation comprising a stabilised complex of CRH and alpha-2 macroglobulin, wherein the formulation is obtainable by the method of any one of claims 1 to 6.

8. A formulation comprising a stabilised complex of corticotropin releasing hormone (CRH) and alpha-2 macroglobulin, wherein the formulation when administered to a patient results in an in vivo CRH expression concentration at 48 hours post-administration that is substantially the same as at 24 hours post-administration.

9. The formulation of claim 7 or claim 8, comprising more than 150,000 pg/ml of alpha-2 macroglobulin. 10. A formulation comprising a stabilised complex of corticotropin releasing hormone (CRH) and alpha-2 macroglobulin, wherein the formulation comprises more than 150,000 pg/ml of alpha-2 macroglobulin.

1 1 . The formulation of any one of claims 7 to 10, comprising more than 250,000 pg/ml or more than 350,000 pg/ml of alpha-2 macroglobulin.

12. The formulation of any one of claims 7 to 1 1 , comprising more than 120 pg/ml of CRH. 13. The formulation of any one of claims 7 to 12, comprising more than 150 pg/ml of CRH.

The formulation of any one of claims 7 to 13, wherein the CRH is non-human.

15. The formulation of any one of claims 7 to 14, further comprising one or more stabilisers.

16. The formulation of claim 15, wherein the one or more stabiliser is selected from fibronectin and albumin.

17. The formulation of any one of claims 7 to 16, further comprising a proopiomelanocortin (POMC) peptide.

18. The formulation of claim 17, wherein the POMC peptide is non-human.

19. The formulation of claim 17 or claim 18, comprising at least 140 pmol/L of POMC peptide.

20. The formulation of any one of claims 7 to 19, further comprising one or more of vasopressin, ACTH, MSH such as a-MSH, β- MSH, and γ-MSH, LPH such as β-LPH and γ-LPH, β-endorphin, enkephalin such as met-enkephalin and leu-enkephalin, CLIP, and Lipotrophin-gamma.

21 . The formulation of any one of claims 7 to 20, further comprising CRH binding protein (CRH-BP).

22. The formulation of claim 21 , comprising less than 50 pg/ml of CRH-BP.

23. The formulation of any one of one of claims 7 to 22, for use in prevention or treatment of one or more diseases selected from Alzheimer's disease; systemic sclerosis (SSc); multiple sclerosis; rheumatoid arthritis; osteoarthritis; optic neuritis; motor neuron disease; hepatitis, in particular hepatitis C; autoimmune diseases including lupus, psoriasis, eczema, thyroiditis, and polymyositis; axonal or nerve damage; cancers, in particular myelomas, melanomas, and lymphomas; neural disorders, both demyelinating and non-demyelinating; Parkinson's disease; inflammatory conditions; inflammatory tendonopathies; inflammatory bowel disease; inflammation of the lung, including emphysema, lung fibrosis, alveolitis and cystic fibrosis; obesity; nerve conduction disorders; and sexual dysfunction, in particular erectile dysfunction.

A method of treatment for a disease selected from Alzheimer's disease; systemic sclerosis (SSc); multiple sclerosis; rheumatoid arthritis; osteoarthritis; optic neuritis; motor neuron disease; hepatitis, in particular hepatitis C; autoimmune diseases including lupus, psoriasis, eczema, thyroiditis, and polymyositis; axonal or nerve damage; cancers, in particular myelomas, melanomas, and lymphomas; neural disorders, both demyelinating and non-demyelinating; Parkinson's disease; inflammatory conditions; inflammatory tendonopathies; inflammatory bowel disease; inflammation of the lung, including emphysema, lung fibrosis, alveolitis and cystic fibrosis; obesity; nerve conduction disorders; and sexual dysfunction, in particular erectile dysfunction; the method comprising administering the formulation of claim 1 1 or the formulation of any one of one of claims 7 to 22 to a patient in need thereof.